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Both Anti-Inflammatory and Antiviral Properties of Novel Drug Candidate Both anti-inflammatory and antiviral properties of novel drug candidate ABX464 are mediated by modulation of RNA splicing Audrey Vautrin, Laurent Manchon, Aude Garcel, Noëlie Campos, Laure Lapasset, Abdelhamid Mahdi Laaref, Roman Bruno, Marie Gislard, Emeric Dubois, Didier Scherrer, et al. To cite this version: Audrey Vautrin, Laurent Manchon, Aude Garcel, Noëlie Campos, Laure Lapasset, et al.. Both anti- inflammatory and antiviral properties of novel drug candidate ABX464 are mediated by modulation of RNA splicing. Scientific Reports, Nature Publishing Group, 2019, 9 (1), 10.1038/s41598-018-37813-y. hal-02867141 HAL Id: hal-02867141 https://hal.archives-ouvertes.fr/hal-02867141 Submitted on 27 May 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License www.nature.com/scientificreports OPEN Both anti-infammatory and antiviral properties of novel drug candidate ABX464 are mediated by Received: 16 August 2018 Accepted: 11 December 2018 modulation of RNA splicing Published: xx xx xxxx Audrey Vautrin2, Laurent Manchon1, Aude Garcel2, Noëlie Campos2, Laure Lapasset2, Abdelhamid Mahdi Laaref1, Roman Bruno3, Marie Gislard4, Emeric Dubois 4, Didier Scherrer2, J Hartmut Ehrlich2 & Jamal Tazi1 ABX464 is a frst-in-class, clinical-stage, small molecule for oral administration that has shown strong anti-infammatory efects in the DSS-model for infammatory bowel disease (IBD) and also prevents replication of the HIV virus. ABX464 which binds to cap binding complex (CBC) has demonstrated safety and efcacy in a phase 2a proof-of-concept clinical trial in patients with Ulcerative colitis. Previously, with limited technologies, it was not possible to quantify the efect of ABX464 on viral and cellular RNA biogenesis. Here, using RNA CaptureSeq and deep sequencing, we report that ABX464 enhances the splicing of HIV RNA in infected PBMCs from six healthy individuals and also the expression and splicing of a single long noncoding RNA to generate the anti-infammatory miR-124 both ex vivo and in HIV patients. While ABX464 has no efect on pre-mRNA splicing of cellular genes, depletion of CBC complex by RNAi leads to accumulation of intron retention transcripts. These results imply that ABX464 did not inhibit the function of CBC in splicing but rather strengthens it under pathological condition like infammation and HIV infection. The specifc dual ability of ABX464 to generate both anti-infammatory miR-124 and spliced viral RNA may have applicability for the treatment of both infammatory diseases and HIV infection. ABX464 is a novel drug candidate for treating patients infected with human immunodefciency virus (HIV) and patients with ulcerative colitis (ABIVAX, data in file). Despite the successful control of viremia, many HIV-infected individuals treated with ART exhibit residual infammation associated with non-AIDS-related morbidity and mortality. Several reports have shown that measures of infammation and immune activation are the best independent predictors of disease progression in HIV-infected individuals. Tus, the anti-infammatory activity of ABX464 is potentially relevant for the intended use in treating HIV patients, in whom the infamma- tion around viral reservoirs was shown to substantially contribute to adverse cardiovascular and tumorigenic efects despite long-term ART-treatment. In addition, ABX464 protects mice from the lethal efects of DSS (Dextran Sulphate Sodium), which is a key animal model for infammatory bowel disease1. Patients with UC may beneft from ABX464 which has demonstrated safety in phase 2 clinical trial (ABIVAX, data in fle) and has a mode of action diferent from classical medications including corticosteroids, immunomodulators and biologic treatments. ABX464 is a small molecule that binds to the cap binding complex (CBC)2, a complex at the 5′-end of the pre-mRNA transcript that promotes the initial interaction with transcription and processing machinery3–5. Te CBC recruits several factors to m7G-modifed transcripts to mediate processing events and is required for efcient cellular and viral pre-mRNA splicing3. Te interaction of CBC with the U1 snRNP at the 5′ splice site of the frst intron in the transcript4,6 and direct interaction of CBC with proteins in U4/U5/U6 particles enhances the formation of spliced mRNAs5,7. Although CBC is not essential for viability in either yeast or humans8,9, its deletion results in a reduction in the recruitment of several splicing factors to the nascent transcript, 1IGMM, CNRS, Univ. Montpellier, Montpellier, France. 2ABIVAX, 1919 route de Mende, 34293, Montpellier Cedex 5, Montpellier, France. 3ACOBIOM, 1682 Rue de la Valsière, 34184, Montpellier Cedex 4, Montpellier, France. 4MGX, Univ Montpellier, CNRS, INSERM, Montpellier, France. Audrey Vautrin and Laurent Manchon contributed equally. Correspondence and requests for materials should be addressed to J.T. (email: [email protected]) SCIENTIFIC REPORTS | (2019) 9:792 | https://doi.org/10.1038/s41598-018-37813-y 1 www.nature.com/scientificreports/ resulting in inhibition of cotranscriptional spliceosome assembly5. Te CBC complex has also been shown to afect microRNA biogenesis10–12. miRNAs are transcribed by RNA pol II as primary (pri)-miRNAs, which carry the m7G cap13. During nuclear and cytoplasmic processing events, the pri-miRNA loses the m7G cap, and the mature, 21–23-nucleotide-long miRNA is incorporated into RISC (RNA-induced silencing complex) to guide RNA silencing12. Since a large fraction of miRNA genes are located in introns14,15, the CBC complex may be involved in the interplay between the processing of intronic pre-miRNAs and pre-mRNAs16,17. ABX464 inhibits viral replication by afecting the biogenesis of viral RNA2 but its efect on cellular and viral RNA biogenesis has not been analyzed in detail. ABX464 will only act on viral replication once proviral DNA was integrated to cellular DNA. Tis is important as the viral genome, once integrated in infected cells, requires both activation and inhibition of precursor mRNA splicing18,19. Successful infection and production of new infectious HIV particles requires the balanced expression of seven viral proteins (Rev, Tat, Nef, Vif, Vpr, Vpu and Env) that are produced by splicing of the HIV-1 primary 9 kilobases (kb) transcript; among these, the Tat and Rev factors are essential for viral gene expression at the transcriptional and posttranscriptional levels in infected cells18,19. Te HIV-1 primary transcript serves not only as genomic RNA for progeny virus but also as the mRNA that encodes the viral Gag and Gag-Pol proteins18,19. While most cellular unspliced RNAs are retained in the nucleus, where they are degraded20, nuclear export of the unspliced viral RNAs is facilitated by the Rev protein through binding to the Rev responsive element (RRE)21–24 and interaction with CRM1-dependent export machinery23,25. Terefore, inefcient alternative splicing is required to maintain a balance between HIV gene expression and viral production18,26. Tis balance is thought to be mediated by the HIV long terminal repeat (LTR) and the presence of suboptimal viral 5′ and 3′ splice sites (5′ and 3′ ss), which are positively regulated by regulatory sequences and their recognition by cognate trans-acting cellular factors26–32. By binding the CBC complex, ABX464 has been shown to interfere with Rev-mediated export of unspliced RNA2. However, the underlying mechanisms behind modulation of viral and cellular splicing and/or miRNA biogenesis by the binding of ABX464 to CBC are pres- ently unknown. In this study, we elucidate the mechanism of action of ABX464 as small molecule to treat patients with HIV infection and IBD. Using RNA capture and deep sequencing we demonstrate that both efects are mediated through the same mechanism i.e. enhanced pre-mRNA splicing. ABX464 not only enhances pre-mRNA splicing of HIV viral RNA to block HIV replication but also triggers the splicing of a long non-coding RNA which houses one of the loci for the anti-infammatory miR-124 and thereby increases the expression of the anti-infammatory microRNA, miR-124. Our fndings may open up new treatment options for patients with HIV and infammatory diseases. Results ABX464 generates spliced HIV RNA variants. To profle viral transcriptional events modulated by ABX464 and thereby assess the full depth of the HIV transcriptome, we employed a recently described targeted RNA capture and sequencing strategy (RNA CaptureSeq). Tis strategy involves the construction of tiling arrays across the HIV genome, against which cDNAs are hybridized, eluted and sequenced. RNA CaptureSeq is sim- ilar to previous in-solution capture methods33 and exome sequencing approaches34, but when combined with deep-sequencing technology, provides saturating coverage and permits the robust assembly of rare and unanno- tated HIV transcripts35. PBMCs from 6 HIV-negative donors were infected with the YU-2 strain, followed by treatment with ABX464. Our protocol resulted in mild infection that did not lead to cell death at 8 days postinfection (dpi), and viral rep- lication was inhibited by more than 70% with 5 µM of ABX464 (Fig. 1B). Afer cDNA capture from infected cells that were untreated or treated with ABX464, libraries were prepared and sequenced using an Illumina sequencing facility. To highlight potential new splicing events induced by ABX464, we used a custom bioinformatics pipeline with the main step involving the assembly of putative transcripts from targeted RNA sequencing reads to con- struct contigs (Illumina paired-end 2 × 75 bp) (Fig. S1A,B). Te putative transcripts (contigs) were then mapped to the HIV genome.
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